Borrelia burgdorferi is the causative agent of Lyme disease in North America and is transmitted by ticks of the genus Ixodes. It is a highly invasive spirochete that can cause infection and manifestations in humans and other mammals that persist for months to years. The disease has localized, disseminated, and chronic stages, and B. burgdorferi appears to cause dermal, neurologic, cardiovascular, and arthritic symptoms primarily though the ability to invade almost any tissue, establish long-term infection, and induce inflammatory responses. The bacterium produces no known toxins, and its mechanisms of pathogenesis are largely unknown. Genetic studies using low-passage, infectious B. burgdorferi have been challenging due to exceedingly low transformation rates and plasmid loss;as a result, only 10 genes have been investigated to date with regard to their importance in the mammal-tick infectious cycle. In this project, signature-tagged mutagenesis in a transformable, infectious clone of B. burgdorferi B31 will be used to systematically analyze the roles of the 1740 protein-encoding genes in the infection of C3H/HeN mice and Ixodes scapularis ticks. In Aim 1, a library of -3,600 signature-tagged mutants will be constructed and insertion sites analyzed;this library will be made available to all investigators and will serve as a basis for infectivity analysis and the estimation of the minimal gene content required for in vitro growth. In Aim 2, groups of the mutants obtained in Aim 1 will be inoculated into mice and multiple organ sites analyzed for the presence of organisms 2 to 4 weeks after inoculation. Mutants with decreased infectivity will be characterized further by plasmid analysis and gene complementation to determine whether the mutated genes are virulence determinants. Aim 3 will be focused on the identification of genes required for the infectivity, persistence and transmission of B. burgdorferi in Ixodes scapularis ticks. The result of this study will be a comprehensive view of the B. burgdorferi genes required for mammal-tick infectious cycle, and will fuel the detailed analysis of the functional roles of these genes in future years. Lyme disease, the most common arthropod-borne disease in the United States, is caused by the spiral-shaped bacterium Borrelia burgdorferi and related organisms. These bacteria are transmitted by ticks and cause a long-term infection in people that affects the skin, nervous system, joints, and heart. Because we know so little about how B. burgdorferi causes disease, it is difficult to design better ways to prevent, diagnose, and treat Lyme disease. The goal of this study is to identify every B. burgdorferi gene that is important in the infection process, so that we can use the resulting information to help reduce the impact of Lyme disease on people in the United States and in other parts of the world.